Separation of bile acids



?atented Dec. 1, 1953 No Drawing." Application June 13,- 1951, Serial No. 231,439

8' claims: (01. z'soms'u)" "ll 'iis' invention relates as tli s'fi'arjati'ofi dfbil'e acids. flhe invention iarticfularlvus'ffil' the separation of cholic acid from the total. acid pie t well known, d'soxycliolic acid offiis 1 Xy'ie'ne and many other ofanic crinsadistic coordination cdr'finofids'wliich arusef' and in acfiaifii'ost' unavoidable, int ara on and-purification of; ds'oxvcl'iolic acid iron l natural} sources. Most 6f" theavailabl methods for? the; isolation off deso'iycholi'cf acid involve" the co inec'ipitationof 'cho dacid ariddelsoiich lid acid; followed by the irtrac'tionof the" desj' ycholic acid from-the n'iu'cl'i iargr'cnane acid frac ti'onbyi rile use' ofs'uitable' solventsifri which deoli caicf Has a greater solfibiliti'f 5511 does 2 an by fi 1tration,;c holic acid can also be recipitated without other handling than the adjustment of hydrogen-ionconcentration. H

I acl ieve this result dissolving the cr ude mixed bile acids in aqueous alkalinelsol utii at a concentration prierably in the range: at 15 25% solids. To this solution of bile acid s is added about 5-25% ofan alcohol or ketone together with sumcient non 'polar, volatile organic compound to form a coordination compound with the destmych oli c acid presentin tlie mixture. Acid is" then added slowly and with vigorous agitation to approziirnately pH 6.0 or until cris tab lization has started, whichever occurs first. Acidification is discontinued until the pH has; come to equilibrium due to crystallization of the des xycholic acid coordination compound which is removing hydrogen ions from solution. Further acidification produces little change in the hydrog'en-ion concentration, once the crystallization is well under wa since the precipitating. bile acid is removing hydrogen-ion approximately: as fast as it is added. Addition of minerar acid is continued s Icing as he pH remains" constant and from 0.1 to 012' pH unit beyond that plateau 1'ev'e1 to; insure complete precipitation of the desoxy cl ioli' acid coordination compound. The crystalline material is removed by filtration, den trifugation or other suitable: means and the mother liquor treated with additional acid to precipitate the cholic acid from the solution.

The cholic acid fraction shows a behavior similar to that exhibitedby the desoxycholic; the pH level remainingstable during the crystallization ofthe'cholic acid. By discontimiirig' acidifi' cation a few tenths" (.1 to 12 pH) of one pI-I"unit beyond the plateau level or" the cholic acid, it is possible also to leave' in s'oliition almost all the iiiipuri-ties. r

v In adjusting the pI-f the" abovefstebsgjriiinial acid of other suitable ac'id rnay be used; v

The non-' pola'r; volatile organic substances addedfor forming a coordination compound with the des'oxycholicacid in the miitu're mayb' benzene, toluene, xylene, monobhloibbiizfeii, the dichlorobnzeiies', and man aliii'liat'ic hy'dio carbons; Since such non-polar substances are well known in the art for producing such 06- accomp'anyi-ng tarry impurities and fattyacid' 3 ordination compounds, a further listing of these is believed unnecessary.

Specific examples of the process may be set out as follows:

Example 1 500 kilograms crude mixed bile acids are dissolved in 2500 liters of water by the addition of 50 kilograms of sodium hydroxide. To this solution is added 250 liters of tertiary butyl alcohol and 100 liters of xylene. The temperature of the mixture is adjusted to 45 C. and sulphuric acid is added to pH 6.3 with vigorous agitation. The mixture is seeded by the addition of a small amount of crude desoxycholic acid-xylene coordination compound to induce crystallization of the desoxycholic acid fraction. Agitation is continued with cooling to room temperature until pH equilibrium is reached at pH 6.8. Slow acidification with sulphuric acid is continued at room temperature until the mixture is maintained at.v

acids, and bases are polar substances, while benezne, xylene, toluene, chlorobenzene, dichlorobenzene and the volatile aliphatic hydrocarbons are non-polar substances. As above stated, nonpolar substances are well known in connection with their ability to form coordination compounds with desoxycholic acid.

While the foregoing specification I have set out specific steps and treating substances in considerable detail for the purpose of illustrating embodiments of the invention, it will be understood that such details may be varied widely by those skilled in the art without departing from the spirit of my invention.

I claim:

1. In a process for separating desoxycholic acid from an aqueous alkaline solution of cholic and desoxycholic acids in which a non-polar volatile organic substance is added to form a coordination compound with the desoxycholic acid in the solution, the steps of acidifying the mixture until pH 6.6. The crystalline disoxycholic acid after cooling is separated in a basket centrifugation.

The mother liquid is warmed to 50 C. and acidifi I cation with sulphuric acid continued with agitation to pH 5.8. Crystallization is induced by seeding and pH equilibrium established at pH 5.9. The mixture is cooled to room temperature and acidification with sulphuric acid is resumed and continued to pH 5.6. The precipitate of cholic "acid hydrate is collected by filtration or other means.

Example 2 200 kilograms crude desoxycholic acid-xylene coordination compound, containing approximately cholic acid, is dissolved in 600 liters of water by the addition of 20 kilograms of sodium hydroxide. To this solution is added '10 liters of tertiary butyl alcohol and 20 liters of xylene. The temperature of the mixture is adjusted to C. and sulphuric acid is added with vigorous agitation to pH 7.3. Crystallization takes place at this pH and continues without further addition of acid, the pH meanwhile drifting to 7.4. After pH equilibrium has been established at this level, additional sulphuric acid is added slowly and with continued mixing until the mixture is maintained at pH 7.0. The suspension of desoxycholic acid compound is cooled to room temperature and the solids collected by centrifugation. The mother liquor is warmed to C. and is adjusted to pH 4, permitting the cholic acid to crystallize. The mixture is cooled to room temperature and the cholic acid hydrate collected by filtration.

Example 3 The process was carried through as described in Example 2, except that methyl iso-butyl ketone was employed instead of tertiary alcohol, and comparable results were obtained.

Example 4 The process of Example 1 was carried out, except that benzene was substituted by xylene, and comparable results obtained.

The water-soluble salts of cholic and desoxycholic acids may be formed by adding alkalis salts such as sodium, potassium or lithium hydroxides, by adding ammonia or ammonium hydroxide, etc.

By non-polar is meant a non-electrolyte and an organic compound, the atoms of which are held in electromagnetic union by sharing a common pair of electrons. Thus alcohol, ketones,

crystallization of the desoxycholic coordination compound begins, maintaining the pH against further substantial drop until the bulk of the desoxycholic acid crystallizes, and separating the crystallized desoxycholic acid.

2. In a process for separating desoxycholic acid from an aqueous alkaline solution of cholic and desoxycholic acids in which a non-polar volatile organic substance is added to form a coordination compound with the desoxycholic acid in the solution, the steps of acidifying the mixture until crystallization of the desoxycholic coordination compound begins, continuing the addition of acid r until an equilibrium of the pH is established and until the pH is from about .1 to about .2 below the equilibrium level, and separating the resulting crystallized desoxycholic acid.

3. In a process for separating desoxycholic and cholic acids from a mixture thereof in which from about 5 to 25% of an agent selected from the group consisting of alcohol and ketone is added to an aqueous solution of water-soluble salts of cholic and desoxycholic acids and in which a nonpolar volatile organic substance is added to form a coordination'compound with the desoxycholic acid in the mixture, the steps of acidifying the mixture until crystallization of the desoxycholic acid coordination compound begins, and maintaining the pH of the mixture against further substantial drop until the bulk of the desoxycholic acid crystallizes. 4. The process of claim' 3, in which acid is added after crystallization of the desoxycholic coordination compound begins until an equilibrium is established and until the pH is from .1 to about .2 below the equilibrium level, and separating the resulting crystallized desoxycholic acid.

5. The process of claim 4, in which after the separation of desoxycholic acid, acidification is continued until crystallization of the cholic acid begins, and then maintaining the pH against further substantial drop until the bulk of the cholic acid is crystallized.

6. The process of claim 4, in which after the separation of the desoxycholic acid, acidification is continued until crystallization of the cholic acid begins, and then after a pHvequilibrium has been established, continuing the acidification until the pH reaches about .1 to ".2 below said equilibrium level, and finally removing the cryscholic acid and separating the desoxychol'i'c acid crystals.

ROBERT H. SIFFERD.

References Cited in the file of this patent UNITED STATES PATENTS Number Name Date 2,346,239 Sifferd Apr. 11, 1944 FOREIGN PATENTS Number Country Date 105,769 Great Britain Dec. 11, 1919 582,772 Great Britain Nov. 27, 1946 

1. IN A PROCESS FOR SEPARATING DESOXYCHOLIC ACID FROM AN AQUEOUS ALKALINE SOLUTION OF CHOLIC AND DESOXYCHOLIC ACIDS IN WHICH A NON-POLAR VOLATILE ORGANIC SUBSTANCE IS ADDED TO FORM A COORDINATION COMPOUND WITH THE DESOXYCHOLIC ACID IN THE SOLUTION, THE STEPS OF ACIDIFYING THE MIXTURE UNTIL CRYSTALLIZATION OF THE DESOXYCHOLIC COORDINATION COMPOUND BEGINS, MAINTAING THE PH AGAINST FURTHER SUBSTANTIAL DROP UNTIL THE BULK OF THE DESOXYCHOLIC ACID CRYSTALLIZES, AND SEPARATING THE CRYSTALLIZED DESOXYCHOLIC ACID. 